Treatment of diarrhea with strains of Bifidobacterium longum

ABSTRACT

The present invention pertains to novel microorganisms of the genus  Bifidobacterium , that are useful in preventing diarrhea brought about by pathogenic bacteria. In particular, the present invention relates to the use of said microorganisms for the preparation of an ingestable support and to a composition containing the same.

The present invention pertains to novel microorganisms of the genusBifidobacterium, that are useful in preventing diarrhea brought about bypathogenic bacteria. In particular, the present invention relates to theuse of said microorganisms for the preparation of an ingestable supportand to a composition containing the same.

Organisms that produce lactic acid as a major metabolic component havebeen known since long. These bacteria may be found in milk or in milkprocessing factories, respectively, living or decaying plants but alsoin the intestine of man and animals. These microorganisms, summarizedunder the term “lactic acid bacteria”, represent a rather inhomogeneousgroup and comprise e.g. the genera Lactococcus, Lactobacillus,Streptococcus, Bifidobacterium, Pediococcus etc.

Lactic acid bacteria have been utilized as fermenting agents for thepreservation of food taking benefit of a low pH and the action offermentation products generated during the fermentative activity thereofto inhibit the growth of spoilage bacteria. In addition lactic acidbacteria have been used for preparing a variety of different foodstuffsuch as cheese, yogurt and other fermented dairy products from milk.

Quite recently lactic acid bacteria have attracted a great deal ofattention in that some strains have been found to exhibit valuableproperties to man and animals upon ingestion. In particular, specificstrains of the genus Lactobacillus or Bifidobacterium have been found tobe able to colonize the intestinal mucosa. Their temporary or sustainedmaintenance in the gut has been assumed to have numerous positiveeffects on the health of the beings having incorporating them.

In this respect, EP 0 768 375 discloses specific strains of the genusBifidobacterium, that are capable to become implanted in the intestinalflora and may adhere to intestinal cells. These Bifidobacteria arereported to assist in immunomodulation, being capable to competitivelyexclude adhesion of pathogenic bacteria to intestinal cells, thusassisting in the maintenance of the individual's health.

During the last few years research has also focused on the potential useof lactic acid bacteria as probiotic agents. Probiotics are consideredto be viable microbial preparations which promote the individual'shealth by preserving the natural microflora in the intestine. Amicrobial preparation may be commonly accepted as a probiotic in casethe effectual microbes thereof and their mode of action are known.Probiotics are deemed to attach to the intestine's mucosa, colonize theintestinal tract and likewise prevent attachment of harmfulmicroorganisms thereon. A crucial prerequisite for their action residesin that they have to reach the gut's mucosa in a proper and viable formand do not get destroyed in the upper part of the gastrointestinaltract, especially by the influence of the low pH prevailing in thestomach.

In this respect, WO 97/00078 discloses a specific strain, termedLactobacillus GG (ATCC 53103), as such a probiotic. The microorganism isparticularly employed in a method of preventing or treating food inducedhypersensitivity reactions in that it is administered to a recipienttogether with a food material that has been subjected to a hydrolysistreatment with pepsin and/or trypsin. The Lactobacillus strain selectedis described as exhibiting adhesive and colonizing properties andshowing a protease enzyme system, so that the protein material containedin the foodstuff to be administered is further hydrolyzed by means ofproteases secreted by the specific Lactobacillus strain. The methoddiscussed in this document shall eventually result in the uptake ofprotein material by the gut that does not show a substantial amount ofallergenic material anymore.

Further, in EP 0 577 903 reference is made to the use of such lacticacid bacteria having the ability of replacing Heliobacter pylori, theacknowledged cause for the development of ulcer, in the preparation of asupport intended for the therapeutic or prophylactic treatment of anulcer associated with the action of Heliobacter pylori.

In view of the valuable properties particular strains of lactic acidbacteria may provide, there is a desire in the art for additional lacticacid bacterial strains that are beneficial to the well being of manand/or animal.

Consequently, a problem of the present invention is to provideadditional bacterial strains that exhibit new properties beneficial forman and/or animals, such as pets.

The above problem has been solved by providing novel microorganismsbelonging to the genus Bifidobacterium having the capability ofpreventing colonization of the intestine with pathogenic bacteriacausing diarrhea and using them for the preparation of an ingestablesupport material.

The Bifidobacterium is selected from the group consisting ofBifidobacterium longum CNCM I-2169 and Bifidobacterium longum CNCMI-2170.

The microorganisms of the present invention have been shown to exhibitinter alia the following properties: they are gram positive, catalasenegative and CO₂ production negative, they produce L(+) lactic acid andmay essentially prevent colonization of intestinal cells by bacteriabringing about diarrhea, such as pathogenic E. coli, e.g.enteropathogenic E. coli (EPEC), or salmonella, e.g. Salmonellatyphimurium.

The novel microorganisms may be used for the preparation of a variety ofingestable support materials, such as e.g. milk, yogurt, curd, fermentedmilks, milk based fermented products, fermented cereal based products,milk based powders, infant formulae and pet food and may be included inthe support in an amount of from about 10⁵ cfu/g to about 10¹¹ cfu/g.For the purpose of the present invention the abbreviation cfu shalldesignate a “colony forming unit” that is defined as number of bacterialcells as revealed by microbiological counts on agar plates.

The present invention also provides a food or a pharmaceuticalcomposition containing at least one of the above Bifidobacteria and/orcontaining a supernatant, in which the microorganisms have been grown oran active fraction thereof, respectively. In this respect it has beenfound that the supernatant of such bacteria also exhibits ananti-pathogenic activity.

For preparing a food composition according to the present invention atleast one of the Bifidobacteria of the present invention is incorporatedin a suitable support, in an amount of from about 10⁵ cfu/g to about10¹² cfu/g, preferably from about 10⁶ cfu/g to about 10¹⁰ cfu/g, morepreferably from about 10⁷ cfu/g to about 10⁹ cfu/g.

In case of a pharmaceutical preparation the product may be prepared inform of tablets, liquid bacterial suspensions, dried oral supplements,wet oral supplements, dry tube feeding or a wet tube feeding with theamount of the Bifidobacterium/Bifidobacteria to be incorporated thereinbeing in the range of up to about 10¹² cfu/g, preferably from about 10⁷cfu/g to about 10 ¹¹ cfu/g, more preferably from about 10⁷ cfu/g toabout 10¹⁰ cfu/g.

The activity of the novel microorganisms in the individual's intestineis of course dose dependent. That is, the more the novel microorganismsare incorporated by means of ingesting the above food material or thepharmaceutical composition the higher the protective and/or curingactivity of the microorganisms. Since the novel microorganisms are notdetrimental to mankind and animals and have eventually been isolatedfrom baby feces a high amount thereof may be incorporated so thatessentially a high proportion of the individual's intestine will becolonized by the novel microorganisms.

Yet, according to another embodiment the supernatant of a culture of aBifidobacterium of the present invention, or an active fraction thereof,may be used for preparing the ingestable support. The supernatant may beused as such or may be dried under conditions that do not destroy themetabolic compounds secreted by the micro-organisms into the liquidmedium, such as e.g. freeze drying, and may be included in the carrier.In order to minimize the number of unknown compounds in the supernatantthe Bifidobacteria will preferably be grown in a defined media, thecomposition of which is known and does not negatively affect the hostincorporating it. Further, the skilled person will, based on his generalknowledge optionally deplete the supernatant from unwanted products,such as e.g. by means of chromatography.

In the figures,

FIG. 1 shows a graph, indicating the capability of the cell lines CNCMI-2169 (termed B128/Cal) and Bifidobacterium longum CNCM I-2170 (termedBL29/F9) to adhere to human intestinal cells in culture.

FIG. 2 shows the pathogen sensitivity of pathogenic bacteria towardsBifidobacterium longum CNCM I-2170 (BL29/F9).

FIG. 3 shows the pathogen sensitivity of pathogenic bacteria towardsBifidobacterium longum CNCM I-2169 (B128/Cal).

FIG. 4 shows the activity of the cell lines B128/Cal and BL29/F9 againstS. typhimurium SL1344 infecting Caco-2 cells.

FIG. 5 shows the rate of survival of mice infected with Salmonellatyphimurium SL 1344 and treated with the Bifidobacterium BL29/F9.

During the extensive studies leading to the present invention theinventors have investigated baby feces and isolated a variety ofdifferent bacterial strains therefrom. These strains were subsequentlyexamined for their capability to prevent colonization and/or invasion ofepithelial cells with bacteria that are known to cause diarrhea, such asE. coli, Sigella, Klebsiella, Yersinia, Pseudomonas aeruginosa Listeria,Streptococcus, Staphilococcus, Clostridium difficile, H. pyori and alsoCandida albicans.

Several bacterial genera comprising Bifidobacterium, Lactococcus andStreptococcus were screened for their diarrhea inhibitory properties.The tests for the inhibitory property were performed with pathogenicmicroorganisms, such as E. coli, Klebsiella, Yersinia, Pseudomonasaeruginosa, H. pyori, and Salmonella typhimurium as representatives forpathogenic microorganisms causing diarrhea in affected individuals.

The various bacteria were grown in a suitable medium, such as MRS,Hugo-Jago or M17 medium at temperatures of from about 30 to 40° C.corresponding to their optimal growth temperature. After reachingstationary growth the bacteria were collected by centrifugation andresuspended in physiological NaCl solution. Between the different teststhe bacterial cells were stored frozen (−20° C.).

For assessing anti-bacterial properties the following approaches werechosen.

According to one protocol cultured Bifidobacteria of the presentinvention were examined for their capability to decrease the viabilityof the different pathogenic microorganisms. To this end, a culture ofpathogenic bacteria was contacted with a concentrated supernatant of aBifidobacterium culture and the growth potential of the pathogenicbacteria was assessed.

According to a second protocol the adhesion capability of theBifidobacteria of the present invention to T₈₄ cells, a cell culturemodel for the intestine, was determined. To this end, the Bifidobacteriawere cultured with T₈₄ cells and the rate of adhesion was assessed.

According to another protocol the potential of the Bifidobacteria of thepresent invention to prevent infection of intestinal cells bySalmonella, using the cell line Caco-2 as a model for the intestine, wasdetermined. In this respect, the supernatant of a cell culture of theBifidobacteria of the present invention was added together with thepathogenic microorganism to the intestinal cells and the rate ofadhesion, or invasion, respectively, was assessed.

Thus, it could be shown that cultured Bifidobacteria and the supernatantproofed to be extremely effective in preventing both adhesion to andinvasion into the intestinal cells indicating that metabolic compoundssecreted by the novel microorganisms are likely to be responsible forthe anti-diarrhea activity.

The present invention will now be described by way of examples withoutlimiting the same thereto.

Media and solutions:

-   MRS (Difco)-   Hugo-Jago (tryptone 30 g/l (Difco), yeast extract 10 g/l (Difco),    lactose 5 g/l-   (Difco), KH₂PO₄ 6 g/l, beef extract 2 g/l (Difco), agar 2    g/l/(Difco))-   M17 (Difco)-   Eugon Tomato Agar (Canned tomato juice 400 ml, Eugon agar BBL 45.5    g, Maltose Difco-   10 g, Hemin Sigma 5 mg, Agar Difco 5 g, distilled water 600 ml)-   DMEM (Dulbecco's modified Eagle medium)-   CFA (according to Ghosh et al. Journal of Clinical Microbiology,    1993 31 2163–6)-   Müller Hinton agar (Oxoid)-   LB (Luria Bertami, Maniatis, A Laboratory Handbook, Cold Spring    Harbor, 1992)-   C¹⁴-acetate (53,4 Ci/mMol, Amersham International PLC)-   PBS (NaCl 8 g/l, KCl 0.2 g/l, Na₂HPO₄ 1.15 g/l, KH₂PO₄ 0.2 g/l))-   Trypsin-EDTA solution (Seromed)-   FCS Fetal calf serum (Gibco)

E. coli DAEC C 1845 was obtained from Washington University, Seattle andE. coli JPN15 was obtained from the Center for Vaccine Development ofthe University of Maryland, USA).

The Salmonella typhimurium strain SL1344 was obtained from thedepartment of Microbiology, Stanford University, CA, USA. This strainacts as a pathogen on mice and is resistant to Streptomycin. It adheresto Caco-2 colon cells (Finlay and Falkow, 1990).

The Klebsiella was obtained from stock clinical isolates from themicrobiological laboratory of the Faculté de Pharmacie Paris XI,Châtenay-Malabry, France.

The Yersinia was obtained from INSERM Unit 411, Hôpital Necker, Paris,France.

The Pseudomonas aeruginosa was obtained from stock clinical isolatesfrom the microbiological laboratory of the Facultê de Pharmacie ParisXI, Châtenay-Malabry, France.

The H. pylori was obtained from Institute of Microbiology, LausanneUniversity, Lausanne, Switzerland.

EXAMPLE 1

Isolation of Bifidobacteria

Fresh feces were harvested from diapers of 16 healthy babies 15 to 27days old. 1 g of fresh feces was placed under anaerobic conditions fortransportation to the laboratory and microbiological analyses were runwithin 2 hours from sampling by serial dilutions in Ringer solution andplating on selective media. Eugon Tomato Agar (Canned tomato juice 400ml, Eugon agar BBL 45.5 g, Maltose Difco 10 g, Hemin Sigma 5 mg, AgarDifco 5 g, distilled water 600 ml) incubated anaerobically at 37° C. for48 hours was used to isolate bifidobacteria. Colonies were randomlypicked up and purified. Physiological and genetic characterisation wasperformed on the isolates.

EXAMPLE 2

Cultivating Cell Lines

Caco-2 Cells:

For the inhibition assays the cell line Caco-2 was utilized as a modelof mature enterocytes of the small intestine. This cell line presentscharacteristic of intestinal cells such as e.g. polarization, expressionof intestinal enzymes, production of particular structural 30polypeptides etc. The cells were grown on different supports, namely onplastic dishes (25 cm², Corning) for growth and propagation, on defattedand sterilized 6 well glass plates (22×22 mm, Corning) for the adhesionand the inhibition tests. After the second day in culture the medium(DMEM) was changed on a daily basis. Before use the medium wassupplemented with 100 U/ml penicilline/streptomycine, 1 μg/mlamphoterine, 20% FCS inactivated at 56° C. for 30 min and 1% of asolution containing non-essential amino acids (10 mM) (Eurobio, Paris,France). Culturing was performed at 37° C. in an atmosphere comprising90% air and 10% CO₂. The cells were splitted every six days. The cellswere detached from the walls of the well by treatment in PBS with 0.015%trypsine and 3 mM EDTA at pH 7.2. For neutralizing the effect oftrypsine an equal volume of the culture medium containing FCS was addedto the cell suspension obtained, the mixture was centrifuged (10 min at1000 rpm) and the pellet was again dissolved in culture medium. Livingcells (not dyed with trypane blue) were counted. About 3.5×10⁵ livingcells were transferred to a new culture bottle and about 1.4×10⁵ cellsper well and cultivated until a confluent monolayer was obtained.

T₈₄ Cells:

For the adhesion assays the cell line T₈₄ was utilized as a model ofcolon cells from the intestine. This cell line presents characteristicsof intestinal cells such as e.g. polarisation, expression of intestinalenzymes, production of particular structural polypeptides etc. T₈, cellswere obtained from University of California, San Diego, Calif. Cellswere grown in DMEM (50%) and Ham's F12 (50%) supplemented with 2 mMglutamine, 50 mM HEPES, 1% non-essential amino acids and 10% inactivated(30 min, 56° C.) fetal calf serum (Boehringer, Mannheim, Germany) at 37°C. in a 10% CO₂/90% air atmosphere. Cells were seeded at a concentrationof 10⁶ cells per cm². Cells were used for adherence assays at latepost-confluence, i.e., after 10 days.

All strains except Bifidobacteria were kept at −80° C. in their culturemedium containing 15% glycerol. As the number of transfers into newmedia has an influence on the adhesion factors, the Salmonella strainwas only transferred twice within a period of 24 hours, the firsttransfer taking place when the strain was frozen. All cultures wereraised aerobically.

Bifidobacteria

The bacterial strains (Bifidobacterium longum CNCM I-2169 (B128/Cal) andBifidobacterium longum CNCM I-2170 (BL291/F9)) were stored at −20° C. inMRS medium containing 15% glycerol. The strains were grown underanaerobic conditions in MRS and transferred twice to new media atintervals of 24 hours before use in the inhibition assays. For the assaya concentration of 2×10⁹ cfu/ml was utilized. The supernatant wascollected by centrifugation for 1 hour at 20.000 rpm and the supernatantobtained was subsequently checked for the presence of bacteria. Thestrains of Bifidobacterium were cultivated anaerobically in MRS during18 hours at 37° C. The cultures were then centrifuged (20 min. at 4°C.), the supernatant was collected, lyophilized, returned to thesolution and then concentrated ten times (10×). The pH of thesupernatant was finally adjusted to 4.5.

E. coli C 1845:

The first passage after thawing was effected on a CFA—Müller Hintonagar, which is suitable to effect expression of adhesion factors by thebacterium. Before each experiment the bacterial cells were incubated at37° C. with a transfer to a new medium being effected twice after 24hours each.

Klebsiella:

Bacteria were grown overnight for 18 hrs at 37° C. in Luria broth.

Yersinia:

Bacteria were grown overnight for 18 hrs at 37° C. in Luria broth.

Pseudomonas aeruginosa:

Bacteria were grown overnight for 18 hrs at 37° C. in Luria broth.

H. pylori:

Bacteria were grown on Brain-Heart Infusion (BHI)-agar plates containing0.25% yeast extract (Difco Laboratories, Detroit, Mich.), 10% horseserum and 0.4% Campylobacter selective complement (Skirrow supplement,SR 69; Oxoid Ltd, Basingstoke, England).

EXAMPLE 4

Adhesion of B128/Cal and BL29/F9 to T₈₄ and Caco-2 Cells

The Caco-2 and T₈₄ monolayers, prepared on glass coverslips which wereplaced in six-well Corning tissue culture plates (Corning Glass Works,Corning, N.Y.), were washed twice with phosphate-buffered saline (PBS).Bifidobacteria (1 ml, 4×10⁸ bacteria/ml in spent culture supernatant,treated-supernatant or fresh MRS broth) were added to 1 ml of the cellline culture medium. This suspension (2 ml) was added to each well ofthe tissue culture plate and the plate incubated at 37° C. in 10%CO₂/90% air. After 1 hour of incubation, the monolayers were washed fivetimes with sterile PBS, fixed with methanol, stained with Gram stain,and examined microscopically. Each adherence assay was conducted induplicate over three successive passages of intestinal cells. For eachmonolayer on a glass coverslip, the number of adherent bacteria wasevaluated in 20 random microscopic areas. Adhesion was evaluated by twodifferent technicians to eliminate bias.

The results are shown in FIG. 1 from which it becomes obvious that bothof B128/Cal and BL29/F9 are capable to adhere to intestinal cellscomparable to the known cell line GG (WO 97/00078) or Lal (EP 0 577903).

EXAMPLE 5

Anti-Pathogenic Activity of the Bifidobacteria

As candidates for pathogenic bacteria E. coli, Klebsiella, Yersinia,Pseudomonas aeruginosa and H. pyori were used.

Based on a culture of bacteria (B128/Cal or BL29/F9) kept in MRS mediumfor 18 hours, an exponentially growing culture was produced (3 hours at37° C.). 2 ml of this solution were removed and centrifuged for 5 min.at 5500 g, +4° C. After collection of the supernatant the cell pelletwas washed in sterile PBS. After centrifuging, the pellet was collectedand 2 ml of sterile PBS were added. The bacteria were counted and thesuspension was adapted in such a way that between 1 and 5×10⁶bacteria/ml were produced.

The assessment of the antimicrobial effect exerted by the Bifidobacteriaof the present invention was carried out according to the Lehrer methoddescribed in Lehrer et al., J. Imunol. Methods 137 (1991), 167–173,which document is incorporated here by way of reference. The resultsthereof are shown in FIGS. 2 and 3.

From the above results it may be seen that the Bifidobacteria of thepresent invention may effectively inhibit growth of the variouspathogenic bacteria.

EXAMPLE 6

Inhibition Assay for Salmonella

Salmonella are bacteria that invade epithelial cells and multiplytherein. For determining the inhibitory activity of the Bifidobacteriaof the present invention towards Salmonella typhimurium the strain SL1344 and following procedure was used.

The pathogenic cells were cultivated in LB-medium. After the secondpassage to new medium the bacterial strains were marked withradioisotopes using C¹⁴-acetate at 10 μCi/ml in LB-medium. Incubation ofthe strains in this medium was performed for 18 hours at 37° C.

The bacterial suspension was subsequently subjected to centrifugation(1041 rpm, 15 min) so as to eliminate the remaining C¹⁴-acetate from thesupernatant. The pellet was suspended and washed in PBS and the cellswere suspended at a concentration of about 10⁸ cells/ml in 1% sterilemannose. Mannose is known to inhibit non specific adhesion. Thebacterial solution was then adjusted to 2×10⁸ cells/ml.

The pathogen (1 ml; 2×10⁸ cells) and an aliquot of a supernatant (1 ml)of a Bifidobacterium culture are pre-incubated for 2 hours at 37° C. Thesuspension is subsequently centrifuged, the resulting supernatant isremoved and the pellet is again suspended in 0.5 ml PBS. This pathogensolution (0,5 ml) is then brought in contact with human intestine cellsin culture.

The culture was washed with sterile PBS twice and 0,5 ml adhesion medium(DMEM) was added. The cells are then incubated for 1 hour at 37° C.under 10% CO₂.

After incubation the number of bacteria in the incubation medium andon/in the intestinal cells are counted. In order to determine the amountof cells adhering on or having invaded into the intestinal cells thefollowing approaches have been chosen.

For determining the number of adhering bacteria the medium was decantedand the cells were washed once with culture medium and once with sterilePBS. Subsequently, 1 ml of sterile H₂O was added per compartment, tolyse the cells and to form a cell solution which was incubated for 1–2hours at 37° C., after which successive dilutions were carried out. Inorder to count the number of adhering and invasive bacteria, the cellsolution was centrifuged to remove cell debris and the radioactivity wasmeasured.

According to another protocol 10 aliquots were each put on TSA medium.The media were then incubated for 18–24 hours at 37° C.

For determining the amount of invaded bacteria the Caco-2 cells werewashed with PBS so as to eliminate all non-adhering cells. Subsequently,a medium containing gentamycin (20 μg/ml) was added and incubation wascontinued for 1 hour at 37° C. Gentamycin is an antibiotic notpenetrating intestinal cells so that all extracellular microorganismswere killed, while bacteria having already invaded intestinal cells willsurvive. The cells were then incubated for another hour at 37° C. andwere then washed twice with PBS. The cells were lysed by addition of andincubation in sterile distilled water for for 1–2 hours at 37° C. Afterremoving the cell debris radioactivity was determined. According toanother protocol successive dilutions were carried out, which were puton TSA medium. Incubation: 18–24 hours at 37° C.

It may be seen that cultured cells and the culture supernatant wereextremely effective in preventing adhesion of and invasion intointestinal cells by Salmonella.

EXAMPLE 7

Infection of Mice by the Strain S. typhimurium C5

Adult, 7–8 weeks old, axenic, female mice (C3H/He/oujco conventional,Iffa Credo, France), raised under sterile conditions, were orallyinfected with a fixed concentration of S. typhimurium (0,2 ml, 10⁸cfu/mouse). Some mice were rendered monoxenic by the implantation of arange of Bifidobacteria strains. With some mice, the Bifidobacteria insegments of the intestine were counted after its removal and mincing ofthe organs in PBS. With other mice, the protection against infection wasassessed in such a way that they were continuously kept in a sterileenvironment and the days of survival were compared to the control group.

The results are shown in FIG. 5. As may be derived therefrom in thecontrol group nearly all mice died after a time period of about 10 days.In contrast thereto, all mice treated with BL29/F9 were alive after 10days with only 20% dying from the detrimental effect exerted bySalmonella after a period of 30 days.

These results show the extreme superior properties of the Bifidobacteriaof the present invention.

1. A method of treating diarrhea, the method comprising administering toa mammal at risk of diarrhea a composition including a therapeuticallyeffective amount of a Bifidobacterium capable of preventing colonizationof bacteria causing diarrhea wherein the Bifidobacterium is selectedfrom the group consisting of Bifidobacterium longum CNCM I-2169 andBifidobacterium longum CNCM I-2170.
 2. The method according to claim 1wherein the amount of Bifidobacterium administered ranges from about 10⁵cfu/g to about 10¹² cfu/g.
 3. The method according to claim 1 whereinthe composition is administered as a food composition selected from thegroup consisting of milk, yogurt, curd, fermented milk, a milk basedfermented product, a fermented cereal product, a milk based powder, aninfant formula and a pet food.
 4. The method according to claim 1wherein the composition is administered as a pharmaceutical composition.5. A method of treating diarrhea, the method comprising administering toa mammal at risk of diarrhea a composition including a therapeuticallyeffective amount of a Bifidobacterium capable of preventing colonizationof bacteria causing diarrhea wherein the composition includes asupernatant of the Bifidobacterium selected from the group consisting ofBifidobacterium longum, CNCM I-2169 and Bifidobacterium longum CNCMI-2170 wherein the Bifidobacterium ranges in concentration from about1×10⁶ cells/ml to about 5×10⁶ cells/ml.
 6. The method according to claim5 wherein the amount of Bifidobacterium administered ranges from about10⁵ cfu/g to about 10¹² cfu/g.
 7. The method according to claim 5wherein the composition is selected from the group consisting of a foodcomposition and a pharmaceutical composition.